USPTO Patents Application 09993647

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

per

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 5

C07D 498/22, A61K 31/335
C07H 19/23 // (C07D 498/223
C07D 307:00, 273:00, 209:00
C07D 209:00, 209:00)

A2

(11) International Publication Number:

(43) International Publication Date:

WO 94/04541

3 March 1994 (03.03.94)

(21) International Application Number: PCT/US93/07054

(22) International Filing Date : 30 July 1 993 (30.07.93)

(30) Priority data:

07/929,199

12 August 1992 (12.08.92) US

(60) Parent Application or Grant

(63) Related by Continuation
US

Filed on

07/929,199 (CIP)
11 August 1992(11.08.92)

(71) Applicant (for all designated States except US): THE UP-
JOHN COMPANY [US/US]; 301 Henrietta Street, Kal-
amazoo, MI 49001 (US).

(72) Inventors; and

(75) Inventors/ Applicants (for US only) : ABRAHAM, Irene
[US/US]; 1805 Waite, Kalamazoo, MI 49008 (US).
ARISTOFF, Paul, A. [US/US]; 1650 Brookmoor Lane,
Portage, MI 49002 (US). SKULNICK, Harvey, I. [US/
US]; 1745 Old Deer Run, Kalamazoo, MI 49009 (US).

(74) Agent: WOOTTON, Thomas, A; The Upjohn Company,
301 Henrietta Street, Kalamazoo, MI 49001 (US).

(81) Designated States: AT, AU, BB, BG, BR, BY, CA, CH,
CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, LK,
LU, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU,
SD, SE, SK UA, US, VN, European patent (AT, BE,
CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL,
PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
GN, ML, MR, NE, SN, TD, TG).

Published

Without international search report and to be republished
upon receipt of that report.

(54) Title: PROTEIN KINASE INHIBITORS AND RELATED COMPOUNDS COMBINED WITH TAXOL

(57) Abstract

This invention describes both known and novel compounds, some of which are protein kinase inhibitors, that may be com-
bined with taxol type compounds. The combination of disclosed compounds plus taxol type compounds exhibits powerful syner-
gistic effects and the combinations are useful in the treatment of cancer. The novel compounds and their synthesis are described.
A compound of formula (I), above, is described wherein Ri-Rg represent various substituents.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

AT

Austria

FR

France

MR

Mauritania

AU

Australia

CA

Gabon

MW

Malawi

BB

Barbados

CB

United Kingdom

NE

Niger

BE

Belgium

CN

Guinea

NL

Netherlands

BF

Burkina Faso

CR

Greece

NO

Norway

BG

Bulgaria

HU

Hungary

NZ

New Zealand

BJ

Benin

IE

Ireland

PL

Poland

BR

Brazil

IT

Italy

PT

Portugal

BY

Belarus

JP

Japan

RO

Romania

CA

Canada

KP

Democratic People's Republic

RU

Russian Federation

CF

Central African Republic

of Korea

SD

Sudan

CG

Congo

KR

Republic of Korea

SE

Sweden

CH

Switzerland

KZ

Kazakhstan

SI

Slovenia

CI

Cote d'lvoire

LI

Liechtenstein

SK

Slovak Republic

CM

Cameroon

LK

Sri Lanka

SN

Senegal

CN

China

LU

Luxembourg

TD

Chad

cs

Czechoslovakia

LV

Latvia

TC

Togo

cz

Czech Republic

MC

Monaco

UA

Ukraine

DE

Germany

MG

Madagascar

US

United States of America

DK

Denmark

ML

Mali

oz

Uzbekistan

ES

Spain

MN

Mongolia

VN

Viet Nam

FI

Finland

WO 94/04541 PCT/US93/07054

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PROTEIN KINASE INHIBITORS AND RELATED
COMPOUNDS COMBINED WITH TAXOL
FIELD OF THE INVENTION

This invention describes the use of compounds that are used in combination with taxol
5 to control cancerous growths and tumors. Protein kinase inhibitors and related compounds are
combined with taxol and taxol related compounds and the combination of compounds exhibits
powerful potentiating effects when used to treat cancer. Many of the compounds are protein
kinase inhibitors, other compounds achieve similar effects but are not necessarily protein kinase
inhibitors.

10 BACKGROUND OF THE INVENTION

Taxol was first isolated from the bark of the western yew, Taxus brevifolia, and
identified as an antitumor agent in 1971 by Wani, et al. Recently, phase n clinical trials with
taxol have shown it to be one of the most exciting chemotherapeutics available. Taxol has
proven effective in drug-refractory ovarian cancer (McGuire, et al., 1989), and has shown a 56%
15 objective response rate in metastatic breast cancer (Holmes, et al., 1991). In addition, there is
reason for hope that taxol may be effective in many other types of cancers.

The development of taxol, however, has faced many obstacles. Taxol's poor solubility
required that it be administered in the vehicle Cremophor EL (polyethylated castor oil), which
led to a high incidence of hypersensitivity reactions. It is not clear whether these reactions are
20 caused by the vehicle or the drug, but it was found that using longer drug infusions (Weiss, et
al., 1990) and anti-allergic regimens (Rowinsky, et al., 1990) reduced the incidence of such
reactions. In addition, there are inherent problems in producing sufficient quantities of taxol.
Extraction from the bark of the extremely slow growing western yew using present methods
cannot meet the demand for taxol. Cultivation of the western yew may take years to establish,
25 synthesis of the complex taxol molecule will be difficult andVor very expensive. Alternative
sources of taxol or a taxol substitute or a taxol additive would therefore be highly desirable.

Taxol has been shown previously to be toxic to tumor and leukemia cells inoculated in
mice, including L-1210, P388 and P-1534 leukemia cells and Walker WM-256 carcinosarcoma,
sarcoma 180 and Lewis lung tumor cells (Wani et al., 1971). It has also been shown to be toxic
30 to cultured human HeLa cells (Schiff et al., 1979 ) and CHO (Chinese hamster ovary) cells

(Cabral et al., 1981). This evidence of toxicity to rodent and human tumor cells in vitro and to
tumor bearing mice in vivo predicted that taxol would be an active chemotherapeutic agent and
led to clinical trials in human cancer patients. These clinical trials showed efficacy of taxol in
treating ovarian cancer (McGuire et al., 1989). Taxotere is a taxol type compound that has also
35 been shown to have powerful antitumor activity. Bissery et. al, Cancer Research 51, 4845-4852,
Sept. 15, 1991.

WO 94/04541 PCT/US93/07054

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Since taxol is now known to be an effective chemotherapeutic agent, a co-treatment that
increases the toxicity of taxol on cancer or transformed cells, such as CHO cells, would be
likely to increase the chemotherapeutic effect of taxol in cancer patients or to allow smaller
doses of taxol to be administered. Quantities of taxol available are extremely limited.
5 Compounds that increase the efficacy of taxol, thereby allowing smaller amounts to be used
with equal effectiveness, will enable more patients to be treated with taxol. This should also
reduce the hypersensitivity and non-therapeutic toxic reactions seen clinically with taxol, as both
less taxol and the less of the vehicle needed to deliver taxol will need to be administered.

Our finding is that when the compounds of this invention are combined with taxol or
10 taxol related compounds the mixture of compounds has a potentiating effect that, surprisingly
produces tumor cell toxicity at lower doses than taxol alone. These findings, and other studies,
suggest that the compounds will be effective in synergizing with taxol in killing tumor cells in
human cancer patients. The findings also suggest results may be seen with taxol related
compounds such as taxotere and related taxol analogues.
15 INFORMATION DISCLOSURE

Many of the compounds of this invention are related to the physiologically active
substance K-252. The following patents disclose some of these compounds. U.S. 4,877,776
issued October 31, 1989. U.S. 4,923,986 issued May 8, 1990. W.O 8807-045-A published
September 22, 1988.

20 The following Japanese patent applications also disclose related compounds: J63 295-

588-A, J63 295-589-A, J62 155-284-A, and J62 155-285-A disclose stausporine related
compounds.

SUMMARY OF THE INVENTION

This invention is in two parts. Known compounds are listed in part I, they are claimed
25 for the method of using the compounds as described herein. The known compounds are also

claimed as compounds combined in a composition with taxol type compounds. The new

compounds are in part II. The new compounds are claimed as compounds, for their method of

use and in a composition.

I. The known compounds.
30 A. Indolocarbazole Type Compounds

1) The "First Known Derivatives of K-252." The "First Known Derivatives
of K-252" are all of the compounds disclosed in U.S. patent 4, 877,776. U.S. patent 4, 877,776
incorporated herein by reference.

2) The "Second Known Derivatives of K-252." The "Second Known

35 Derivatives of K-252" are all of the compounds disclosed in U.S. patent 4,923,986. U.S. patent
4,923,986 incorporated herein by reference.

WO 94/04541 PCT/US93/07054

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3) The specific compounds below are more preferred,
3(a) KT5823

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PCI7US93/07054

3(d) KT5720

5

(il 2 ) 3
CH 2

3(e) Staurosporine
B. Non-Indolecarbazole Type Compounds

1)

Adriamycin

2)

Amiloride

20

3)

Calphostin

4)

Chlorpromazine

5)

The compound known as "HA- 1004"

6)

Indomethacin

7)

Okadaic acid

25

8)

Phenazocine

9)

Polymyxin B

10)

2-aminopurine

11)

6-dimethyl-aminopurine

12)

Sphingosine

30

13)

Tamoxifen

14)

Compounds related to tamoxifen such as triphenylethylene antiestrogens

15)

Trifluoperazine

16)

Verapamil

17)

3-isobutyl- 1 -methyl-xanthine

35

18)

8-Cl-cAMP

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PCT/US93/07054

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II. The new compounds.

A compound of FORMULA I, below,

FORMULA I

wherein,

Rj is -H, -(C r C 4 alkyl), -C(0)-(C r C 4 alkyl), -NH 2 ,
-C(0)-NH 2 , -CH 2 CH2-N(R 1 . 1 )2,
15 wherein R^ is -H or -(Cj-C^ alkyl,

R 2 is -H, or R 2 and R 3 taken together are (O),

R 3 is -H, -OH or R 2 and R 3 taken together are (O),

R 4 is -H, -OH, -NH 2 , or -0-(C r C 4 alkyl),

R 5 is -OH, -0-(C r C 4 alkyl), or -0-C(0)-(C r C 4 alkyl),
20 R 6 is -(C 6 -C 12 alkyl), -(C 3 -C 10 cycloalkyl), -(CH 2 ) n CH 2 N(R 6 _ 1 ) 2 ,

wherein R 6 _j is -H, or -(Cj-C 4 alkyl),

R 7 is -H, or -NH 2 ,

R 8 is -CI, -Br, -H, -CH 3 , -CH 2 OH, -OH,' -0-(C r C 4 alkyl),
-N(R g .!) 2 , or -NHC(0)-NH(R g . 1 ),
25 wherein R g _j is -H or -(Cj-C 4 alkyl)

wherein n is 0-5
with the proviso that:

a) when R 2 or R 3 is -OH then Rj is H,

b) when Rj, R 2 , R 3 , R 4 , and R 7 all equal H and R 5 is OH
30 then R 6 does NOT equal -(CH 2 ) 5 CH 3 ,

c) when Rj, R 4 , and R 7 all equal H, and R 2 combined with R 3 is (O),

and R 5 is OH , then Rg does NOT equal -(CH 2 ) 5 CH 3 .

d) when R 4 is -OH, -NH 2 , or -0(Cj-C 4 alkyl), then R 4 and R g are the same.

35 A pharmaceutical composition consisting of a pharmaceutical ly acceptable carrier and an

effective amount of FORMULA I. A pharmaceutical composition consisting of a

WO 94/04541 PCT/US93/07054

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pharmaceutically acceptable carrier and an effective amount of the compound of FORMULA I
in conjunction with an appropriate dose of taxol or taxol related compounds. A method of
controlling cancerous growths in mammals which comprises administering a therapeutic or
prophylactic dosage of any of the three following groups of compounds in conjunction with an
5 appropriate dose of taxol or taxol related compounds. 1) a compound of FORMULA I, 2) any
one of the compounds described in the specification as "Indolecarbazole Type Compounds.", 3)
any one of the compounds described in the specification as "Non-Indolecarbazole Type
Compounds."

BRIEF DESCRIPTION OF THE FIGURES

10 Figure 1. Isobologram showing potentiating effect of the combination of Taxol

plus KT5823. The isobologram shows the effectiveness of a combination of 2 drugs for the
killing of wild type, 10001a CHO, cells. The data line is the solid line with open circle or
triangle data points. The data line shows the combination of doses which gives an LD 50 for the
cells. The diagonal dashed line shows the predicted concentrations of drugs if their combination

15 only had an additive effect. If any data points were above the dashed line that would indicate
the combination of compounds had antagonistic effects. Data points below the line indicate the
compounds have potentiating or synergistic effects.

Figure 2. Isobologram showing potentiating effect of the combination of taxol plus
KT5926.

20 Figure 3. Isobologram showing potentiating effect of the combination of taxol plus

KT5720.

Figure 4. Isobologram showing NO potentiating effect from the combination of
taxol plus H-9. This isobologram shows the predicted effect of a "control" substance that does
NOT act in a potentiating or synergistic manner.
25 Figure 5. Isobologram showing potentiating effect of the combination of taxol plus

K252a.

Figure 6. Isobologram showing potentiating effect of the combination of taxol plus
tamoxifen.

Figure 7. Isobologram showing potentiating effect of the combination of taxol plus
30 2-aminopurine.

Figure 8. Isobologram showing potentiating effect of the combination of taxol plus
6-dimethylaminopurine.

Figure 9. Isobologram showing potentiating effect of the combination of taxol plus
chlorpromazine.

35 Figure 10. Isobologram showing potentiating effect of the combination of taxol plus

3-isobutyl- 1 -methyl-xanthine.

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Figure 1 1. Isobologram showing potentiating effect of the combination of taxol plus
8-Cl-cAMP.

Figure 12. Isobologram showing potentiating effect of the combination of taxol plus
Example A-l.

5 Figure 13. Isobologram showing potentiating effect of the combination of taxol plus

Example B-l.

Figure 14. Isobologram showing potentiating effect of the combination of taxol plus
Example B-2.

Figure 15. Isobologram showing potentiating effect of the combination of taxol plus
10 Example B-3.

Figure 16. Isobologram showing potentiating effect of the combination of taxol plus
Example B-4.

Figure 17. Isobologram showing potentiating effect of the combination of taxol plus
Example B-5.

15 Figure 18. Isobologram showing potentiating effect of the combination of taxol plus

Example B-6.

Figure 19. Isobologram showing potentiating effect of the combination of taxol plus
Example B-7.

Figure 20. Isobologram showing potentiating effect of the combination of taxol plus
20 Example B-8.

Figure 21. Isobologram showing potentiating effect of the combination of taxol plus
Example B-9.

Figure 22. Effect of KT5720 and taxol on the growth of MX-1 tumors.
Figure 23. Table of data showing toxicity of several of the drugs both individually
25 and in combination with taxol on non-tumored mice. (In Vivo Effects)

DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention are of two types. The first type are known compounds
described here for their usefulness when combined with taxol type compounds and used to treat
cancer. The second type of compounds are novel compounds described here for the first time.
30 These novel compounds are also useful when combined with taxol type compounds and used for
the treatment of cancer.

L Known Compounds
I. The known compounds, and the source of those compounds, are listed below, and
described by name and by reference to the labeled structures.
35 A) Indolocarbazole Type Compounds

1) The "First Known Derivatives of K-252." The "First Known Derivatives

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PCT/US93/07054

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of K-252" are all of the compounds disclosed in U.S. patent 4, 877,776. U.S. patent 4, 877,776
incorporated herein by reference.

2) The "Second Known Derivatives of K-252." The "Second Known
Derivatives of K-252" are all of the compounds disclosed in U.S. patent 4,923,986. U.S. patent

5 4,923,986 incorporated herein by reference.

3) The specific compounds below are more preferred,
3(a) KT5823

CH,

10

15

20

25

30

3(b) K-252A

3(c) KT5926

H 3 C

0. /

0=C— 0

H 3 C

0=C

CH,

35

WO 94/04541

PCT/US93/07054

3(e) Staurosporine
15 B. Non-Indolecarbazole Type Compounds

1) Adriamycin s

2) Amiloride s

3) Calphostin s

4) Chlorpromazine s

20 5) The compound known as "HA- 1004"

6) Indomethacin s

7) Okadaic acid

8) Phenazocine s

9) Polymyxin B s
25 10) 2-aminopurine s

11) 6-dimethyl-aminopurine s

12) Sphingosine s

13) Tamoxifen

14) Compounds related to tamoxifen such as triphenylethylene antiestrogens
30 15) Trifluoperazine 8

16) Verapamil 8

17) 3-isobutyl-l-methyl-xanthine s

18) 8-Cl-cAMP

35 Compounds marked with a superscript s are available from Sigma Chemical Company.

Taxol and taxotere can be obtained from The National Cancer Institute.

WO 94/04541 PCI7US93/07054

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The clinical pharmacology of taxol is reviewed by Eric K. Rowinsky and Ross C.
Donehower, The Clinical Pharmacology and Use of Antimicrotubule Agents in Cancer
Chemotherapeutics, Pharmac. Ther., Vol 52, pp 35-84, 1991. Clinical and preclinical studies
with taxol are reviewed by William J. Slichenmyer and Daniel D. Von Hoff, Taxol: A New and
5 Effective Anti-cancer Drug, Anti-Cancer Drugs, Vol. 2, pp 519-530, 1991.

Taxol and analogs thereof are the subject of various patents including, for example, U.S.
Patent Nos. 4,814,470; 4,857,653; 4,942,184; 4,924,011; 4,924,012; 4,960,790; 5,015,744;
5,157,049; 5,059,699; 5,136,060; 4,876,399 as well as PCT Publication No. WO 92/09589,
European Patent Application 90305845.1 (Publication No. A2 0 400 971), 89400935.6
10 (Publication No. Al 0 366 841) and 90402333.0 (Publication No. 0 414 610 Al), 87401669.4
(Al 0 253 739), and PCT Publication Nos. WO 91/17977, WO 91/17976, WO 91/13066, WO
91/13053.

Rebeccamycin is described in: T. Kaneko and H. Wong, Tetrahedron Letters, Vol. 26,
No. 34, pp 4015-4018 (1985).
15 The compounds known as K252a, K252b, KT5720, KT5823, KT5926, okadaic acid and

staurosporine, are available from Kamiya Biomedical Company, Thousand Oaks, California.

The compounds known as "H-7," "H-9" and "HA- 1004" (B4-B6) are available from
Seikagaku America, Inc., St Petersburg, Florida

Lavendustin (B8) is available from Gibco BRL.
20 The compound, Kampferol-7-neonesperidoside, is available from Apin Chemical Co.,

Abingden, Oxfordshire, United Kingdom.

All of the documents referred to above are incorporated by reference herein.

II. New Compounds
II. The new compounds of this invention are identified in two ways: by the descriptive
25 name and by reference to structures contained in appropriate charts. In some situations, the
proper stereochemistry is also represented in the charts.

In this document the parenthetical term (C n -C m ) is inclusive such that a compound of
(Cj-Cg) would include compounds of one to 8 carbons and their isomeric forms. The various
carbon moieties are defined as follows: Alkyl refers to an aliphatic hydrocarbon radical and
30 includes branched or unbranched forms such as methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, and n-
octyl.

Alkoxy as represented by -0-(Cj-Cg alkyl) refers to an alkyl radical which is attached to
the remainder of the molecule by oxygen and includes branched or unbranched forms such as
35 methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy,
isopentoxy, n-hexoxy, isohexoxy, n-heptoxy, isoheptoxy, and n-octoxy.

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(C 3 -Cjo)cycloalkyl refers to a radical of a saturated cyclic hydrocarbon which includes
alkyl -substituted cycloalkyl, such as cyclopropyl, 2-methylcyclopropyl, 2,2-dimethylcyclopropyl,
2,3 diethylcyclopropyl, 2-butylcyclopropyl, cyclobutyl, 2-methylcyclobutyl, 3-propylcyclobutyl,
cyclopentyl, 2,2-dimethylcyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. Each of these
5 moieties may be substituted as appropriate.

It will be apparent to those skilled in the art that compounds of this invention may
contain chiral centers. The scope of this invention includes all enantiomeric or diastereomeric
forms of formula I compounds either in pure form or as mixtures of enantiomers or dia-
stereomers. The therapeutic properties of the compounds may to a greater or lesser degree
10 depend on the stereochemistry of a particular compound.

Both organic and inorganic acids can be employed to form non-toxic pharmaceutically
acceptable acid addition salts of the compounds of this invention. Illustrative acids are sulfuric,
nitric, phosphoric, hydrochloric, citric, acetic, lactic, tartaric, palmoic, methanesulfonic,
ethanedisulfonic, sulfamic, succinic, cyclohexylsulfamic, fumaric, maleic, and benzoic acid.
15 These salts are readily prepared by methods known in the art.

The compounds of this invention can be made in accordance with the processes
described in the PREPARATIONS AND EXAMPLES for the preparation of novel compounds
and illustrated in the GENERAL REACTIONS and the REACTIONS OF CHART A and
CHART B.

20 In clinical practice the compounds of the present invention will normally be

administered by injection, in the form of pharmaceutical preparations comprising the active
ingredient either as a free base or as a pharmaceutically acceptable non-toxic, acid addition salt,
such as the hydrochloride, lactate, acetate, mesylate, methanesulfonate, or sulfamate salt, in
association with a pharmaceutically acceptable carrier. The use and administration to a patient

25 to be treated in the clinic would be readily apparent to a physician or pharmacist of ordinary
skill in the art.

In therapeutical treatment the suitable daily doses of the compounds of the invention
should fall within the following ranges: Taxol, taxotere and related compounds should be
administered from .001 mg/kg to 10 mg/kg, preferably between .05 mg/kg to 5 mg/kg for

30 intravenous administration. The compounds to be combined with taxol should be administered
in the same dosage range. The precise dosage will be apparent to an ordinarily skilled physician
or pharmacologist taking into account factors such as the age, weight, sex, and medical
condition of the patient being treated. Also relevant is the potency of the particular compound
and its ability to potentiate the effects of taxol. The potency of the compounds are indicated by

35 the standard tests described below.
The New Compounds:

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A compound of FORMULA I, below,

5

FORMULA I

10

wherein,

Rj is -H, -(C r C 4 alkyl), -C(0)-(C r C 4 alkyl), -NH 2 ,
-C(0)-NH 2 , -CH 2 CH 2 -N(R 1 . 1 ) 2 ,

wherein Rj.j is -H or -(Cj-C 4 alkyl),
15 R 2 is -H, or R 2 and R 3 taken together are (O),

R 3 is -H, -OH or R 2 and R 3 taken together are (O),

R 4 is -H, -OH, -NH 2 , or -0-(C r C 4 alkyl),

R 5 is -OH, -0-(C r C 4 alkyl), or -0-C(0)-(C r C 4 alkyl),

R 6 is -(C 6 -C 12 alkyl), -(C 3 -C 10 cycloalkyl), -(CH^CH^CR^)^
20 wherein R 6A is -H, or -(Cj-C 4 alkyl),

R 7 is -H, or -NH 2 ,

R g is -CI, -Br, -H, -CH3, -CH 2 OH, -OH, -0-(C r C 4 alkyl),
-NCRg.j)^ or -NHC(0)-NH(R g .!),
wherein Rg.j is -H or -(Cj-C 4 alkyl)
25 wherein n is 0-5

with the proviso that:

a) when R 2 or R 3 is -OH then Rj is H,

b) when Rj, R 2 , R 3 , R 4 , and R 7 all equal H and R 5 is OH

then R 6 does NOT equal -(CH 2 ) 5 CH 3 ,
30 c) when Rj, R 4 , and R 7 all equal H, and R 2 combined with R 3 is (O),

and R 5 is OH , then Rg does NOT equal -(CH 2 ) 5 CH 3 .
d) when R 4 is -OH, -NH 2 , or -0-(C|-C 4 alkyl), then R 4 and R g are the same.

Preferred Compounds

35 The preferred compounds of this invention are those, referring to the compound of

FORMULA I, wherein Rj is H or CH 3 ; R 2 , R 3 , and R y is H;' R 5 is OH or OCH 3 ;

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R g is -0-(C r C 4 alkyl). The following compounds are preferred. Example B-4 and Example
A-1, Example A-1 whose structure is shown below.

(Example A-1)

10

Biological Activity

Since taxol is known to be an effective chemotherapeutic agent, for example in the
15 treatment of ovarian cancer, any co-treatment that increases the toxicity of taxol on cancer cells,
such as CHO cells, would be likely to increase the chemotherapeutic effect of taxol in cancer
patients or to allow smaller doses of taxol to be adniinistered. The compounds of this invention
synergize with taxol to produce tumor cell toxicity at lower doses than taxol alone, this requires
the conclusion that the compounds will be effective in synergizing with taxol in killing tumor

20 cells in human cancer patients. Additional studies that evaluate the compounds effects on
human breast call MX-1 tumors, described below also support this conclusion.

The compounds of this invention are •therefore useful for the same cancers for which
taxol has been shown active, including human ovarian tumors, mammary tumors, and malignant
melanoma, lung tumors, gastric tumors, colon tumors, head and neck tumors, and leukemia.

25 See, e.g., the clinical pharmacology of taxol is reviewed by Eric K. Rowinsky and Ross C.
Donehower, The Clinical Pharmacology and Use of Antimicrotubule Agents in Cancer
Chemotherapeutics, Pharmac. Ther., Vol 52, pp 35-84, 1991. Clinical and preclinical studies
with taxol are reviewed by William J. Slichenmyer and Daniel D. Von Hoff, Taxol: A New and
Effective Anti-cancer Drug, Anti-Cancer Drugs, Vol. 2, pp 519-530, 1991.

30 Cell lines and growth .

The parental CHO line, 10001a, is a subclone of the CHO line Pro" 5 (Stanley et al.,
1975). The line was maintained in alpha-MEM Earle's Salts supplemented with 2 mM
glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin and 10% fetal bovine serum.
All cell lines were maintained at 37°C in 5% C0 2 in a humidified incubator. Periodically, the

35 cell lines were tested for mycoplasma and always found to be free of infection.

Compounds were dissolved in dimethylsulfoxide (DMSO) and then diluted into medium for cell

WO 94/04541 PCI7US93/07054

-14-

growth assays.

Drug synergy experiments - "1000 la" cell lines .

Cells were treated simultaneously with the experimental compound and taxol in 132
different combinations of doses in 96 well plates. The 96-weIl plates were incubated for four
5 days. Cell growth was determined by the development of the colorimetric dye 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described by Mosmann, 1983.
MTT dissolved in PBS at 2 mg/ml was added to the plates already containing growth medium
to give a final concentration of 0.2 mg/ml in each well. Plates were then incubated for 3 hours.
The medium containing MTT ± drug was then aspirated off and 100 ul/well isopropanol

10 acidified with 0.04 N HC1 was added. Plates were shaken for 5 minutes and absorbance was
read at 570 nm on a Bio-tek EL 312e Bio-kinetics microplate reader.

Percent growth for 10001a cells was determined by dividing the absorbance reading at
each drug dilution by the reading in control wells. LD 5Q s for each compound were determined
to be the concentration of drug at which a 50% inhibition in cell growth was obtained.

15 Potentiating effects from the combination of compounds on 10001a cells was determined by
graphing the combinations of drugs which gave LD 5Q s in the form of an isobologram (Kallman,
1987 and Brunden, 1988).

The effectiveness of combinations of compounds with taxol on the killing of wild type,
10001a, cells is shown by means of an isobologram. The compounds of this invention act in a

20 potentiating or synergistic manner with taxol to kill cells with much lower doses in combination
than would be expected if the drugs were merely exhibiting additive effects. This effect is
suprising and unexpected. The isobolograms are displayed as FIGURES 1-21. FIGURES 1-21
demonstrate the effectiveness of combinations of compounds with taxol on the killing of wild
type cells. FIGURE 4 is included in the series to show how a compound with no potentiating

25 effect behaves.

The isobologram shows the effectiveness of a combination of 2 drugs for the killing of
wild type, 10001a CHO, cells. The data line is the solid line with open circle or triangle data
points. The data line shows the combination of doses which gives an LD 50 for the cells. In
FIGURES 1-21 the concentration of taxol is plotted against the concentration of drug. The

30 diagonal dashed line shows the predicted concentrations of drugs if their combination only had
an additive effect If any data points were above the dashed line the date would indicate the
combination of compounds had antagonistic effects. Data points below the line indicate the
compounds have potentiating or synergistic effects. Compare the isobologram in FIGURE 4,
showing NO potentiating effect, to the other isobolograms.

35 In addition to the data provided in the isobolograms the compounds have been tested in

mice. Compound KT5720 has been tested on tumored mice and compounds KT5926 and

WO 94/04541 PCT/US93/07054

-15-

KT5720 have been tested in non-tumored mice.

Drug synergy experiments - MX-1 tumors.

FIGURE 22 shows the effect of KT5720 both separately and in combination with taxol
in tumored mice. Human breast cell MX-1 tumors were implanted subcutaneously as 2 mm
5 cubes in athymic mice. Mice were dosed every day for five days with drugs or vehicle control.
The vehicle used was 2% dimethylacetamide, 10% emulphor, 88% saline. Animals received
12.5 mg/kg taxol (shown in figure as solid circle data points), 25 mg/kg KT5720 (shown in
figure as open triangle data points), 12.5 mg/kg taxol + 25 mg/kg KT5720 (shown in figure as
solid triangle data points), or vehicle alone (shown in figure as open square data points). Tumor

10 burden was measured every two or three days starting with day 5 and volume was calculated.
In FIGURE 22 the size of the tumor in millimeters is plotted against time in days. Eight mice
were used per dose group. Results are graphed with standard errors. The results show that there
was no effect of KT5720 alone on inhibition of growth of the tumor cells. Taxol, at 12.5
mg/kg, has a modest effect on reducing the tumor burden in these mice. The combination of

15 KT5720 plus taxol clearly show a potentiation of the taxol effect by the addition of KT5720. In
summary, KT5720 has no effect by itself, but in combination with taxol, at the dosage tested, it
causes a dramatic inhibition of tumor growth.

Drug synergy experiments - non-tumored mice.

FIGURE 23 shows the effects of compounds KT5926 and KT5720 on non-tumored

20 mice. When the compounds are combined with taxol and then administered to non-tumored
normal mice they show a dramatic amount of toxicity. There was no lethality at the doses
shown when these drugs were given individually. This means that there are strong synergistic
effects with the compounds in vivo. The combination of drugs will be effective in tumor
bearing mice and as a cancer treatment for humans. See FIGURE 23 - IN VIVO EFFECTS.

25 This figure provides a table of data showing the toxicity of several of the drugs combined with
taxol as compared to the individual administration of the drugs on non-tumored mice.

The required synergistically effective amounts (concentrations) will vary depending on
the particular types of individual to be treated taking into consideration various conditions
including age, weight, type of cancer treated, stage of disease, etc. Effective amounts can be

30 readily determined by routine experimentation.

Without further elaboration, it is believed that one skilled in the art can, using the
preceding description, practice the present invention to its fullest extent. The following detailed
examples describe how to prepare the various compounds and/or perform the various processes
of the invention and are to be construed as merely illustrative, and not limitations of the

35 preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize
appropriate variations from the procedures both as to reactants and as to reaction conditions and

WO 94/04541

techniques.

-16-

PCI7US93/07054

PREPARATIONS AND EXAMPLES for the preparation of novel compounds.
GENERAL REACTIONS

10

0

Starting materials

15 Step 1

Starting Materials. The starting materials are obtained by using the procedures
described in U.S. Patent 4,923,986 and U.S. Patent 4,877,776. U.S. patents 4,923,986 and
4,877,776 are incorporated by reference into this document.

In addition to the patent references above, the starting materials for the reactions are also

30 described in non-patent literature. The compound known as K252A is described in Kase, H., K.
Iwahashi, and Y. Matsuda, K252a, "A Potent Inhibitor of Protein Kinase C from Microbial
Origin." /. Antiob. (Tokyo) 39:10066-1071 (1986). The compound known as KT 5926, and
related compounds are described in S. Nakanishi, K. Yamada, K. Iwahasha, K. Kuroda and H.
Kase, "KT5926, a Potent and Selective Inhibitor of Myosin Light Chain Kinase." Molecular

35 Pharmocology, 37:482-488 (1990). The other compounds of formula 1 where R 6 is C r C 5 alkyl
are described in U.S. Patent 4,923,986 and U.S. patent 4,877,776. In general, treatment of

WO 94/04541 PCT/US93/07054

-17-

compounds of formula 1, where R 6 is CH 3 , are treated with R^-OH where (R^ is C 6 -C 12 alkyl)
and KCN to give the desired compounds. All the starting materials are described in the above
patents. Compounds of the type R 5 is H are described in WO 91/09034 published 27 June
1991. All the above documents are incorporated by reference herein.
5 The General Procedure for producing variations for the Rg group is as follows:

To an appropriate starting material such as KT252a add an alcohol such as n-hexanol
(Rg is -(CH 2 ) 5 CH 3 ). Stir the mixture at temperatures ranging from room temperature to 125
degrees until dissolution is complete. An equal weight amount of solid KCN is added and the
reaction mixture is stirred for an additional 1 8 to 144 hours at temperatures ranging from room

10 temperature to 125 degrees. The reaction mixture is poured into ethyl acetate and the ethyl
acetate solution is extracted with water. The organic solution is dried over anhydrous sodium
sulfate, filtered and evaporated to dryness under high vacuum at temperatures ranging from 35°
to 70°C to near dryness. Hexane is added to the residue and the resulting solids are allowed to
sit, under hexane, for 24 hours. The solids are filtered and washed well with hexane and dried

15 at 40°C to give the desired compound. The materials may be identified by their retention time
on HPLC. Detailed HPLC conditions are provided in the examples below. For example, when
the following HPLC conditions are used the retention time for KT252a is 2.54 minutes. HPLC:
Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 watenacetonitrile; 2 ml/minute).

20

WO 94/04541

REACTIONS OF CHART A

-18-

PCT/US93/07054

5

10

20

(KT 5926-A)

0

(Example A-l)

25

Procedure A. Reactions of step 1, above. Preparation of Example A-l, (R g is -O-
(CH 2 ) 2 CH 3 , Rg is -(CH 2 ) 5 CH 3 ), from the starting material KT-5926.
Example A-l is named:

9, 12-Epoxy- 1 H-diindolo( 1 ,2,3-fg:3 \2 ', 1 '-kl)pyrrolo(3,4-i)( 1 ,6)benzodiazocine-
30 10-carboxylic acid, 2,3,9,10,1 1,1 2-hexahydro-10-hydroxy-9- methyl -l-oxo-16-propoxy-, hexyl
ester, (9R-(9.alpha.,10.beta., 12.alpha.)).

To 0.8 mg of KT-5926 (0.0015 mmol) is added 0.5 ml of n-hexanol and, after
dissolution is complete, 1.0 mg of KCN is added. The reaction mixture is stirred at room
temperature for 24 hours at which time HPLC indicates all the starting material has reacted.
35 Add 0.5 ml of a 35:65 wateracetonitrile solution, followed by acetonitrile (to complete

dissolution) and chromatograph the entire reaction solution on a preparatory HPLC system (2-

WO 94/04541 PCT/US93/07054

-19-

PrepPak 25 X 100 mm Cartridge) microBondapak CI 8, 10 microns; 35:65 water:acetonitrile at 8
ml/min taking 24 ml fractions. The fractions containing Example A-l, are combined and
evaporated to dryness to give 0.12 mg of product. HPLC data was run as follows: HPLC: Altex
Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 watenacetonitrile; 2 ml/minute) it is
5 18.69 minutes for Example A-l; it is 3.63 minutes for KT 5926.

WO 94/04541

REACTIONS OF CHART B

-20-

PCT/US93/07054

15 Step 1

(Example B-l)

30 Procedure B. Reactions of step 1, above. Preparation of Example B-l, (R g is H, is

-(CH 2 ) 6 CH 3 ), from the starting material, K252a.
Example B-l is named:

9, 12-Epoxy- 1 H-diindolo(l ,2,3-fg:3 ',2 ', r-kl)pyrrolo(3,4-i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 1,12-hexahydro- 10-hydroxy-9-
35 methyl- 1-oxo-, heptyl ester, (9R-(9.alpha.,10.beta.,12.alpha.)).

To 15 mg of KT252a (0.032 mmol) add 2 ml of n-heptanol (R^ is

WO 94/04541 PCT/US93/07054

-21-

-(CH 2 ) 6 CH 3 ). The mixture is allowed to stir at room temperature until dissolution is complete.
15 mg of KCN is added and the reaction mixture is stirred for an additional 96 hours. The
reaction mixture is poured into 20 ml of ethyl acetate and the ethyl acetate solution is extracted
with water. The organic solution is dried over anhydrous sodium sulfate, filtered and evaporated
5 to dryness under high vacuum at 59°C to near dryness. 15 ml of hexane is added to the residue
and the resulting solids are allowed to sit, under hexane, for 24 hours. The solids are filtered
and washed well with hexane and dried at 40°C to give 7.8 mg of Example B-l. HPLC: Altex
Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 watenacetonitrile; 2 ml/minute) retention
time is 7.14 minutes for Example B-l; 2.54 minutes for K252a. Mass Spec, theory predicts
10 552.2498 mass units; Measured: 552.2484.

Example B-2, (R 6 is -(CH 2 ) 7 CH 3 ), is named:

9,12-Epoxy-lH-dimdolo(l,23-fg:3\2\r-kl)pyrroIo(3,4-i)(l,6)berLzodiazocine-
10-carboxylic acid, 2,3,9, 10, 11,1 2-hexahydro-l 0-hydroxy-9-
15 methyl- 1-oxo-, octyl ester, (9R-(9.alpha.,10.beta.,I2.aIpha.)).

Using procedure B only substituting n-octanol (Rg is -(CH 2 ) 7 CH 3 ) in the reaction
described above, and stirring at room temperature for 120 hours, gives Example B-2, as an
amber solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65
watenacetonitrile; 2 ml/minute) retention time is 3.325 minutes. Mass Spec, theory predicts
20 566.2655 mass units; Measured: 566.2652.

v

Example B-3, (R 6 is -(CH 2 ) 8 CH 3 ), is named:

9, 12-Epoxy- 1 H-diindolo(l ,2,3-fg: 3 ',2 ' , 1 '-kl)pyrrolo(3,4- i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9, 10, 11,1 2-hexahydro-l 0-hydroxy-9-
25 methyl- 1-oxo-, nonyl ester, (9R-(9.alpha.,10.beta.,12.alpha.)).

Using procedure B only substituting n-nonanol (Rg is -(CH 2 ) g CH 3 ) in the reaction
described above, and stirring at room temperature for 144 hours, gives Example B-3, as an
amber solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (25:75
watenacetonitrile; 2 ml/minute) retention time is 6.54 minutes. Mass Spec, theory predicts
30 580.281 1 mass units; Measured: 580.2819

Example B-4, (R 6 is -CH(CH 2 CH 3 )((CH 2 ) 3 CH 3 ), is named:

9, 12-Epoxy- 1 H-diindoIo(l ,2,3-fg:3 \2\ r-kl)pyrrolo(3,4-i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 1,1 2-hexahydro-l 0-hydroxy-9-
35 methyl- 1-oxo-, 1-ethylpentyl ester.

Using procedure B only substituting 3-heptanol (Rg is -CH(CH 2 CH 3 )((CH 2 ) 3 CH 3 ) in

WO 94/04541

PCT/US93/07054

-22-

the reaction described above, stirring at 1 10 degrees for 144 hours, gives Example B-4, as an
amber solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65
watenacetonitrile; 2 ml/minute) retention time is 6.44 minutes. Mass Spec, theory predicts
552.2498 mass units; Measured: 552.2501.

5

Example B-5, (R fi is -CHCH 3 (CH 2 ) 4 CH 3 ), is named:

9, 1 2-Epoxy- 1 H-diindolo( 1 ,2,3-fg:3 ',2 ' , 1 '-kl)pyrrolo(3,4- i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,11, 12-hexahydro-10-hydroxy-9-
methyl-l-oxo-, 2-methylhexyl ester.
10 Using procedure B only substituting 2-heptanol (Rg is -CHCH3(CH 2 )4CH 3 ) in the

reaction described above, and heating at 100 degrees for 18 hours, gives Example B-5, as an
amber solid. HPLC retention time is 6.67 minutes. Mass Spec, theory predicts 552.2498 mass
units; Measured: 552.2501.

15 Example B-6, (R 6 is -CHCItyCH^gCIty, is named:

9, 12-Epoxy-lH-diindolo(l,2,3-fg:3'aM '-kl)pyrrolo(3,4- i)(l ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-
methyl-l-oxo-, 2-methylheptyl ester.

Using procedure B only substituting 2-octanol (P^ is -CHCH 3 (CH2) 5 CH 3 ) in the
20 reaction described above, stirring at 100 degrees for 96 hours, gives Example B-6, as an amber
solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (25:75 watenacetonitrile; 2
mlAninute) retention time is 4.55 minutes. Mass Spec, theory predicts 566.2655 mass units;
Measured: 566.2652.

25 Example B-7, (R 6 is -CHCH 3 (CH 2 ) 6 CH3), is named:

9,12-Epoxy-lH-dimdolo(l,2,3-fg:3'^\l'-kJ)pyrrolo(3,4-i)(l,6)rjenzodiazocine-
10-carboxylic acid, 2,3,9,10, 11, 12-hexahydro-10-hydroxy-9-
methyl-l-oxo-, 2-methyloctyl ester, (9R-(9.alpha.,10.beta.,12.alpha.)).

Using procedure B only substituting 2-nonanol (Rg is -CHCH 3 (CH 2 ) 6 CH 3 ) in the
30 reaction described above, stirring at 100 degrees for 96 hours, gives Example B-7, as an amber
solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (25:75 watenacetonitrile; 2
ml/minute) retention time is 6.12 minutes. Mass Spec, theory predicts 580.281 1 mass units;
Measured: 580.2797.

35 Example B-8, (R 6 is -(CH 2 ) 2 OCH 2 CH 3 ), is named:

9, 1 2-Epoxy- 1 H-diindolo( 1 ,2,3-fg:3 ',2 1 '-kl)pyrrolo(3,4- i)( 1 ,6)benzodiazocine-

WO 94/04541

-23-

PCTVUS93/07054

10-carboxyIic acid, 2,3,9, 10,11, 12-hexahydro-l 0-hydroxy-9-
methyI-l-oxo-,2-ethoxyethyl ester, (9R-(9.alpha.,10.beta.,12.alpha.)).

Substituting ethoxyethanol (R 6 is -(Ct^^OCI^CFLj) in the reaction described above,
and stirring at room temperature for 96 hours, followed by 50 degrees for 24 hours, gives
5 Example B-8, as an amber solid. HPLC: Altex Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm,
(35:65 watenacetonitrile; 2 ml/minute) retention time is 1.54 minutes. Mass Spec, theory
predicts 526.1978 mass units; Measured: 526.1959.

Example B-9, (R 6 is -CH(cyclo-CH 2 )5), is named:
10 9,12-Epoxy-lH-dimdolo(l,2,3-fg:3'^\r-kl)pyrrolo(3,4-i)(l,6)r^nzodiazocine-

10-carboxylic acid, 2,3,9,10,11, 12-hexahydro-10-hydroxy-9-

methyl-l-oxo-, cyclohexyl ester, (9R-(9.alpha.,10.beta.,12.alpha.)).

Substituting cyclohexanol (Rg is -CH(cyclo-CH 2 ) 5 ) in the reaction described above,

stirring at 80 degrees for 18 hours, gives Example B-9, as an amber solid. HPLC: Altex
15 Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 watenacetonitrile; 2 ml/minute) retention

time is 3.95 minutes. Mass Spec, theory predicts 536.2185 mass units; Measured: 536.2169.

WO 94/04541

PCT/US93/07054

-24-

TABLE 1 - IN VIVO EFFECTS

mg/kg Dose

Day of Death
Mouse #1 Mouse #2

Taxol

10.0

5.0

5

KT5926

15.0

5.0

1.0

__

0.1

Taxol + KT5926

10.0 + 15.0

10

11

10

10.0+ 5.0

7

10

10.0+ 1.0

9

9

10.0+ 0.1

9

10

5.0 + 15.0

9

14

5.0+ 5.0

9

10

15

5.0+ 1.0

8

9

5.0+ 0.1

10

11

BDF1 mice

Two mice per dose (mouse #1 and mouse #2)

20

Drugs were given intraperitoneally on days 1-9

* No day of death given means that the mice did not die during the duration of the
experiment

WO 94/04541

PCT/US93/07054

-25-
CLAIMS

1. A compound of FORMULA I, below,

FORMULA I

wherein,

Rj is -H, -(C r C 4 alkyl), -C(0)-(C r C 4 alkyl), -NH 2 ,
-C(0)-NH 2 , -CHjCHj-NCRj.j^;
15 wherein R M is -H or -(C r C 4 alkyl);

R 2 is -H, or R 2 and R 3 taken together are, (O);

R 3 is -H, -OH or R 2 and R 3 taken together are, (O);

R 4 is -H, -OH, -NH 2 , or -0-(C r C 4 alkyl);

R 5 is -OH, -0-(C r C 4 alkyl), or -0-C(0)-(C r C 4 alkyl);
20 R 6 is -(C 6 -C 12 alkyl), -(C 3 -C 10 cycloalkyl),(C r C 5 alkyl)-0-(C r C 5 alkyl),

-(CH 2 ) n CH 2 N(R 6 . 1 ) 2 ;

wherein Rg.j is -H, or -(C r C 4 alkyl);

R 7 is -H, or -NH 2 ;

R 8 is -CI, -Br, -H, -CH 3 , -CH 2 OH, -OH, -0-(C r C 4 alkyl);
25 -N(R 8 . X ) 2 , or -NHCO^-NORg.^;

wherein R g l is -H or -(C r C 4 alkyl);
wherein n is 0-5
with the proviso that:

a) when R 2 or R 3 is -OH then Rj is H;
30 b) when Rj, R 2 , R 3 , R 4 , and R y all equal H and R 5 is OH

then R 6 does NOT equal -(CH 2 ) 5 CH 3 ;

c) when Rj, R 4 , and R 7 all equal H, and R 2 combined with R 3 is (O)

and R 5 is OH , then R 6 does NOT equal -(CH 2 ) 5 CH 3 ;

d) when R 4 is -OH, -NH 2 , or -0-iC r C 4 alkyl), then R 4 and R g are the same.

WO 94/04541 PCI7US93/07054

-26-

2. A compound of claim 1 wherein

Rj is -H, -(C r C 4 alkyl), -C(0)-(C r C 4 alkyl), -C(0)-NH 2 .

3. A compound of claim 1 wherein
5 Rj is H or -CH 3 .

4. A compound of claim 1 wherein
Rj is -CH 2 CH 2 -N(R M ) 2 .

10 5. A compound of claim 1 wherein
R 2 and R 3 are H.

6. A compound of claim 1 wherein
R 5 is -OH.

15

7. A compound of claim 1 wherein
R 5 is -OCH 3 .

8. A compound of claim 1 wherein

20 R 6 is -(C7-C 12 alkyl), -(C 3 -C 10 cycloalkyl), -(CH^CHjNCRg.^.

9. A compound of claim 1 wherein

R 6 is -(C 8 -C 12 alkyl), -(C 3 -C 10 cycloalkyl), -(CH^CHjNCRg.j)^

25 10. A compound of claim 2 wherein
R 2 and R 3 is (O).

11. A compound of claim 2 wherein
Rjis -H, -(C r C 4 alkyl);
30 R 2 and R 3 are H and

R 5 is -OH or -0-(Cj-C 4 alkyl).

35

12. A compound of claim 5 wherein
R 5 is -OH or -0-(C r C 4 alkyl).

WO 94/04541 PCT/US93/07054

-27-

13. A compound of claim 5 wherein

R 8 is -CI, -Br, -H, -CH 3 , -CH 2 OH, -OH, -0-(C r C 4 alkyl),
-N(Rg j) 2 , or -NHC(0)-NHR 8 . 1 .

5 14. A compound of claim 5 wherein
R 8 is -0-(C r C 4 alkyl).

15. A compound of claim 1 1 wherein
Rj is -H;

10 R g is -0-(C r C 4 alkyl).

16. A compound of claim 15 which is the compound named,

9, 12-Epoxy- lH-diindolo(l ,2,3-fg:3',2\ 1 '-kl)pyrrolo(3,4- i)(l,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9- methyl- l-oxo-16-propoxy-, hexyl
15 ester, (9R-(9.alpha.,10.beta., 12.alpha.)). (Example A-l)

17. A compound of claim 1 1 selected from the following named compounds,

a) 9, 12-Epoxy- 1 H-diindolo( 1 ,2,3-fg:3 \2' , 1 '-kl)pyrrolo(3,4-i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-methyl-l-oxo-, heptyl ester,

20 (9R-(9.alpha.,10.beta.,12.alpha.)), (Example B-l)

b) 9,12-Epoxy-lH^imdolo(l,2,3-fg:3\2%l'-ld)pyrrolo(3,4-iXl,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-methyl-l-oxo-,l-ethylpentyl ester,
(Example B-4) or

c) 9,12-Epoxy-lH-diindolo(l,2,3-fg:3',2',l '-kl)pyrrolo(3,4- i)(l,6)benzodiazocine-

25 10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-methyl-l-oxo-, 2-methylhexyI ester
(Example B-5).

18. A compound of claim 11 wherein
Rj is -H;

30 R 6 is -(C 8 -C 12 alkyl) and

R g is -0-(C r C 4 alkyl).

WO 94/04541 PCT/US93/07054

-28-

19. A compound of claim 11 wherein
Rj is -H;

R 6 is -(C 3 -C 10 cycloalkyl) or (C r C 5 alkyI)-0-(C r C 5 alkyl) and
R g is -0-(C r C 4 alkyl).

5

20. A compound of claim 1 1 wherein
Rj is -H;

Rg is -(C 8 -C 12 alkyl) and
R g is H.

10

21. A compound of claim 20 selected from the following named compounds,

a) 9J2-Erx)xy-lH-dimdoIo(l,23-fg:3\2\l'-kl)pynoIo(3,4-i)(l,6)beraodiazocine-
10-carboxylic acid, 2,3,9, 10,ll,12-hexahydro-10-hydroxy-9-methyl-l-oxo-, octyl ester,
(9R-(9.alpha.,10.beta.,12.alpha.)); (Example B-2)
15 b) 9,12-Epoxy-lH-diindolo(l,2,3-fg:3',2M'-kl)pyrrolo(3,4- i)(l,6)benzodiazocine-

10-carboxylic acid, 2,3,9, 10,11, 12-hexahydro-10-hydroxy-9-methyl-l-oxo-, nonyl ester,
(9R-(9.alpha.,10.beta.,12.alpha.)); (Example B-3)

c) 9, 1 2-Epoxy- 1 H-diindolo( 1 ,2,3-fg: 3 \2' , 1 '-kI)pyrrolo(3,4- i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,11, 12-hexahydro-10-hydroxy-9-methyl-l-oxo-, 2-methylheptyl ester,

20 (Example B-6)

d) 9, 12-Epoxy- 1 H-diindolo( 1 ,2,3-fg: 3 ' ,2 ', 1 '-kl)pyrrolo(3,4-i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,11, 12-hexahydro-10-hydroxy-9-methyl-l-oxo-, 2-methyloctyl ester,
(9R-(9.alpha.,10.beta.,12.alpha.)) (Example B-7).

25 22. A compound of claim 1 1 wherein
R, is -H;

R<5 is -(C 3 -C 10 cycloalkyl) or (C r C 5 alkyl)-0-(C r C 5 alkyl) and
R g is H.

30 23. A compound of claim 22 selected from,

a) 9,12-Epoxy-lH-diindolo(l,2,3-fg:3',2',r-kl)pyrrolo(3,4- i)( 1 ,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-methyl-l-oxo-,2-ethoxyethyl ester,
(9R-(9.alpha.,10.beta.,12.aIpha.)); (Example B-8) or

b) 9,12-Erx)xy-lH-dimdolo(l,2,3-fg:3%2\l'-kl)pyrrolo(3,4-i)(l,6)r^nzoojazocine-
35 10-carboxylic acid, 2,3,9,10,1 l,12-hexahydro-10-hydroxy-9-methyl-l-oxo-, cyclohexyl ester,

(9R-(9.alpha.,10.beta.,12.alpha.)) (Example B-9).

WO 94/04541 PCT/US93/07054

-29-

24. A pharmaceutical composition for treating, controlling or preventing cancerous growths,
such as human ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric
tumors, colon tumors, head and neck tumors, and leukemia in mammals and humans, which
comprises administering a therapeutic or prophylactic dosage of a compound of FORMULA I in
5 conjunction with an appropriate dose of taxol or taxol related compounds.

25. A pharmaceutical composition for treating, controlling or preventing cancerous growths,
such as human ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric
tumors, colon tumors, head and neck tumors, and leukemia, in mammals and humans, which

10 comprises adniinistering a therapeutic or prophylactic dosage of any one of the compounds
selected from the list below, in conjunction with an appropriate dose of taxol or taxol related
compounds, a) KT5823, b) K252a; c) KT5926, d) KT5720, e) Staurosporine, f) driamycin, b)
Amilorides, c) Calphostin, d) Chlorpromazine, e) The compound known as "HA- 1004", f)
Indomethacin, g) Okadaic acid, h) Phenazocine, i) Polymyxin, j) 2-aminopurine k) 6-dimethyl-

15 aminopurine, 1) Sphingosine, m) Tamoxifen, n) Compounds related to tamoxifen such as
triphenylethylene antiestrogens, o) Trifluoperazine, p) Verapamil, q) 3-isobutyl-l-methyl-
xanthine, r) 8-Cl-cAMP, in conjunction with an appropriate dose of taxol or taxol related
compounds.

20 26. Use of a therapeutic or prophylactic dosage of a compound of FORMULA I in conjunction
with an appropriate dose of taxol or taxol related compounds for the manufacture of a
medicament for the treatment, control or prevention of cancerous growths, such as human
ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric tumors, colon
tumors, head and neck tumors, and leukemia in mammals and humans.

25

27. Use of a therapeutic or prophylactic dosage of the compounds described in U.S. Patents
4,877,776 or 4,923,986, in conjunction with an appropriate dose of taxol or taxol related
compounds for the manufacture of a medicament for the treatment, control or prevention of
cancerous growths, such as human ovarian tumors, mammary tumors, and malignant melanoma,
30 lung tumors, gastric tumors, colon tumors, head and neck tumors, and leukemia in mammals and
humans.

WO 94/04541

PCT/US93/07054

-30-

28. Use of a therapeutic or prophylactic dosage of any of the compounds selected from the
following: a) KT5823, b) K252a, c) KT5926, d) KT5720, or e) Staurosporine, in conjunction
with an appropriate dose of taxol or taxol related compound for the manufacture of a
medicament for the treatment, control or prevention of cancerous growths, such as human

5 ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric tumors, colon
tumors, head and neck tumors, and leukemia in mammals and humans.

29. Use of a therapeutic or prophylactic dosage of any of the compounds selected from the

following: a)

Adriamycin

10

b)

Amilorides

c)

Calphostin

d)

Chlorpromazine

e)

The compound known as "HA- 1004"

f\
i >

TnHnmpthapin

lilUulilVUIttvUl

15

g)

Okadaic acid

h)

Phenazocine

i)

Polymyxin B

j)

2-aminopurine

k)

6-dimethyl-aminopurine

20

1)

Sphingosine

v m )

Tamoxifen

n)

Compounds related to tamoxifen such as triphenylethylene antiestrogens

o)

Trifluoperazine

P)

Verapamil

25

q)

3-isobutyl-l-methyl-xanthine or

r)

8-Cl-cAMP

in conjunction with an appropriate dose of taxol or taxol related compound for the manufacture
of a medicament for the treatment, control or prevention of cancerous growths, such as human
ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric tumors, colon
30 tumors, head and neck tumors, and leukemia in mammals and humans.

WO 94/04541

PCT/US93/07054

FIGURE 1

1/22

ro

CM
CO

m
i—

|OXDl

SUBSTITUTE SHEET

WO 94/04541

PCI7US93/07054

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 3

3/22

CO

(wrf) joxdi

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

WO 94/04541

PCI7US93/07054

FIGURE 5 5/22

O
r °

CM

(flU) |OXDi

WO 94/04541

PCT/US93/07054

|0XD1

WO 94/04541

PCT/US93/07054

FIGURE 7

7/22

c

•

CL

O

C

E
<
I

CM

(flT/) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

SUBSTITUTE SHEET

WO 94/04541 PCT/US93/07054

FIGURE 9 9/22

(flU) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 10

10/22

- o

(Wii) |oxdi

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 1 1 1 1/22

(flu) |OXDl
SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 12 12/22

(flu) |OXDl
SUBSTITUTE SHEET

WO 94/04541 PCT/US93/07054

FIGURE 13 13/22

(flu) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 14 14/22

O

(flu) ioxdi

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

SUBSTITUTE SHEET

WO 94/04541

PCI7US93/07054

SUBSTITUTE SHEET

WO 94/04541 PCT/US93/07054

FIGURE 17 17/22

SUBSTITUTE SHEET

WO 94/04541

PCI7US93/07054

(^U) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

SUBSTITUTE SHEET

WO 94/0454 1 PCT/US93/07054

FIGURE 20 20/22

(flU) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCI7US93/07054

FIGURE 21 21/22

(flU) |OXDl

SUBSTITUTE SHEET

WO 94/04541

PCT/US93/07054

FIGURE 22 22/22

FIGURE 22. TAXOL PLUS KT5720

ON TUMORED MICE

days

SUBSTITUTE SHEET